We had previously sequenced the exomes of individuals from four families, where at least one member was affected with MBS. We identified a few candidate genes (1-3) per family that had variants segregating with MBS (i.e. in affected family members and not in the non-affecteds). Notably, we had not identified candidate genes in common across two or more of the four families studied. Thus, it became clear that interrogating more homogeneous subgroups of affected individuals based on detailed clinical phenotyping was required given the complexity and heterogeneity of this disorder to identify common genetic and potential environmental factors. In light of this, we established a new NHGRI protocol in 2014 (14-HG-0055, ClinicalTrials.gov ID: NCT02055248; PI: Irini Manoli) dedicated to defining the phenotypes and the genetic factors associated with Moebius syndrome and other congenital facial weakness disorders. This protocol has been partially funded by a competitive UO1 grant awarded in January 2014, 1U01HD079068-03 (coPIs: Jabs, Engle, Manoli, Brooks, Pierpaoli) and a 2-year research grant from the Moebius Syndrome Foundation, awarded in January 2017. Initially, our goal was to enroll 24 participants and their family members each year, for 3 years, from 2014 to 2016. Since the beginning of 2017, our goal has been to enroll 10-12 participants per year, based on the funding from the Moebius Syndrome Foundation. As part of the protocol, the affected individual(s) from each family undergoes standardized multisystem phenotyping at the NIH Clinical Research Center (NIH CC). Specialists in ophthalmology, neurology, audiology, otolaryngology, dentistry, craniofacial surgery, speech pathology and rehabilitation medicine evaluate the study participants, providing deep phenotyping data for each specialty. We have enrolled 72 patients and 96 family members as well as 24 healthy controls for brain imaging data analysis, for a total of 192 subjects. To date, 143/192 participants have visited the NIH CC for full clinical evaluation. Initially, we prioritized families with more than one affected individual, since we believed these would provide a better chance of identifying a causative germline mutation. Since 2017, we have been focusing our efforts on enrolling individuals with apparently sporadic and more classic features of MBS. Under the collaborative agreement between NIH, MSSM and BCH, each of the three teams in the UO1 grant (NIH: Manoli/Brooks/Pierpaoli; MSSM Jabs; BCH: Engle) analyzes the clinical and genetic data. To facilitate this, we have created a database to enable sharing of the clinical phenotype information. The detailed phenotype information has already allowed us to categorize the affected individuals into more clearly defined subgroups or categories, which helps inform and direct the genomic analyses that are currently in progress. To date, we have sequenced the exomes of 145 individuals from 46 families. We have identified more than 130 variants in 44 genes segregating with affection/disease status and thus, of interest for follow-up analyses. The Gabriella Miller Kids First Pediatric Research Program awarded resources (Dr. Engle, BCH; X01HL132377) for whole genome sequencing at the Baylor Sequencing Core of 200 individuals and their family members with MBS or hereditary congenital facial paresis (HCFP). Data generated by this funding are being analyzed in collaboration with Daniel MacArthur at the Broad Institute. Our preliminary analyses have identified non-coding variations in a genomic region that is common to several families that meet a specific subset of diagnostic criteria. Analysis of the ENCODE chromatin state data for a neuroblastoma cell line supports a potential regulatory role for this region and we are currently performing functional assays to determine its relevance in our studies. We also assessed four individuals with Carey-Fineman-Ziter syndrome enrolled through John Carey (University of Utah) and Elizabeth Engle (BCH). Candidate mutations had been previously identified (by Drs. Carey and Engle) in a novel gene (myomaker, MYMK) that was common to all four individuals. In collaboration with Stephen Robertson (University of Otago, New Zealand) and Eric Olson (University of Texas Southwestern) we assessed and confirmed the functional relevance of these mutations in human myoblasts and the zebrafish model system. A manuscript describing the MYMK results was published at Nature Communications. Additionally, we have identified compound heterozygous mutations in STAC3, the gene causing Native-American myopathy, in two non-Native American siblings with a CFZS-like phenotype. Brain imaging studies in patients enrolled in the clinical protocol (10 MBS, 5 HCFP and 15 controls) were analyzed by tensor-based morphometry (TBM), a novel approach developed by co-PI Carlo Pierpaoli (NIBIB). This approach enables the identification of morphological changes of individual white matter pathways in subjects with facial palsy, as compared to healthy age and gender matched control subjects. We identified volumetric reduction in the brainstem (posterior pons, pontine tegmentum), indicating atrophy or hypoplasia of the brainstem at this level. The pontine tegmentum contains the nuclei of cranial nerves VI, VII, and the medial longitudinal fasciculus. Similar abnormalities were also observed for the subset of MBS patients with limb defects. However, HCFP did not show hypoplasia in this region. Functional MRI studies are underway with collaborators at NIMH (Japee Shruti and Leslie Ungerleider) to understand differences in emotion processing in MBS subjects compared to controls. Findings to date suggest there are deficits in fearful emotion processing in subjects with MBS, that are accompanied by reduced connectivity in CNS regions that process expressions such as pSTS (posterior superior temporal sulcus) and amygdala. Lastly, fMRI, DTI and electrophysiology studies, including transcranial magnetic stimulation, are being performed in a subset of MBS subjects to understand the underlying pathology causing their mirror movements. We have obtained autopsy material of an individual with classic Moebius syndrome. Postmortem brain imaging and high-resolution DTI reveals aberrant cranial nerve trajectory and highlights the utility of ex vivo micro-imaging approaches for describing abnormal neuroanatomy in human disorders, which is critical to understanding the pathophysiology of cranial nerve abnormalities in MBS. DTI tractography findings were correlated with detailed histopathology of motor neurons in the affected regions. Small areas of calcification were identified on histology at the predicted location of Cn VI, with gliosis in the region of Cn VII. Aberrant dorsoventral tracts were documented near the expected Cn VI tract. A second autopsy specimen from a subject with classic Moebius syndrome became available in the summer of 2018 and is undergoing similar studies to further validate our findings. We believe this collaborative approach of bringing together expert investigators from multiple sites to combine valuable resources/data, will maximize our capacity to identify germline or somatic genetic causes of the various syndromes associated with facial palsy. Moreover, this collaboration has allowed the generation of a patient registry/clinical database in REDCap, including a total of 516 individuals, 204 affecteds, from 169 families, as well as rich phenotypic and genotypic data that will be deposited in dbGaP (Accession ID phs001383.v1.p1.) for further exploration by other investigators interested in conditions involving facial palsy.